Clioquinol induces autophagy by down-regulation of calreticulin in human neurotypic SH-SY5Y cells.

Clioquinol (CQ) is considered as a promising drug of neurodegenerative diseases. However, the underlying mechanism is unclear. Our previous study has proved that CQ induces S-phase cell cycle arrest through the elevation of intracellular calcium concentration ([Ca2+]i) with high levels of SERCA2. Furthermore, it could induce autophagy in an intracellular calcium independent manner in human neurotypic SH-SY5Y cells. In this study, the involvement of calreticulin (CRT) in autophagy induced by CQ was investigated. Our results illustrated the endoplasmic reticulum (ER) stress induced by CQ and DTT led to the cell death in different manners. DTT, an ER stress positive control, induced UPR accompanied with up-regulation of CRT and apoptosis, while CQ inhibited UPR accompanied with down-regulation of CRT，resulting in autophagy. Then, overexpression of CRT was shown to cause UPR and decrease [Ca2+]i, leading to cell apoptosis and inhibition of S-phase arrest induced by CQ. While the UPR was alleviated and autophagy was further enhanced in CRT deficient cells by using targeted siRNA. Meanwhile, down-regulation of CRT resulted in [Ca2+]i overload and induction of S-phase arrest. Finally, we found that the effect of CQ on the HT22 cells was similar to that on the SH-SY5Y cells. Our data showed for the first time that CQ decreased expression of CRT, leading to autophagy, an increase of [Ca2+]i, and cell S-phase arrest in the neurotypic cells. The present study describes the cellular signal pathways regulating autophagy by CQ and highlights the potential therapeutic application of CQ in neurodegenerative disorders.

Clioquinol inhibits cell growth in a SERCA2-dependent manner.

Clioquinol has been reported to act as a potential therapy for neurodegenerative diseases and cancer. However, the underlying mechanism is unclear. We have previously reported that clioquinol induces S-phase cell cycle arrest through the elevation of calcium levels in human neurotypic SH-SY5Y cells. In this study, different types of cells were observed to detect if the effect of clioquinol on intracellular calcium levels is cell type-specific. The Cell Counting Kit-8 assay showed that clioquinol exhibited varying degrees of concentration-dependent cytotoxicity in different cell lines, and that the growth inhibition caused by it was not related to cell source or carcinogenesis. In addition, the inhibition of cell growth by clioquinol was positively associated with its effect on intracellular calcium content ([Ca2+ ]i ). Furthermore, the elevation of [Ca2+ ]i induced by clioquinol led to S-phase cell cycle arrest. Similar to our previous studies, the increase in [Ca2+ ]i was attributed to changes in the expression levels of the calcium pump SERCA2. Comparison of expression levels of SERCA2 between cell lines showed that cells with high levels of SERCA2 were more sensitive to clioquinol. In addition, analysis using UALCAN and the Human Protein Atlas also showed that the expression of SERCA2 in the corresponding human tissues was similar to that of the cells tested in this study, suggesting potential in the application of clioquinol in the future. In summary, our results expand the understanding of the molecular mechanism of clioquinol and provide an important strategy for the rational use of clioquinol.

Clioquinol induces S-phase cell cycle arrest through the elevation of the calcium level in human neurotypic SH-SY5Y cells.

Clioquinol is recently considered to be the most promising drug for treating cancer and neurodegenerative diseases. However, its mode of action varies from different disease models. In this study, we found that clioquinol inhibited cell growth in human neurotypic SHSY-5Y cells, which was attributed to both S-phase cell-cycle arrest and autophagic cell death. Clioquinol increased the intracellular contents of iron and zinc as well as calcium as measured by ICP-AES. Staining of Fluo-3 confirmed an increase in the level of calcium. Analysis of the metal-binding ability of clioquinol showed that it was not a chelating agent of calcium ions and the elevation of intracellular calcium content is not achieved by clioquinol as an ionophore. CaCl2 could simulate or even aggravate the cytotoxicity of clioquinol and it increased S-phase cell cycle arrest induced by clioquinol in a concentration dependent manner. Staining of acridine orange demonstrated that autophagy induced by clioquinol was not affected by addition of calcium ions. In contrast, the intracellular calcium ion chelator BAPTA-am abolished the clioquinol-induced S phase arrest and reduced the cell death caused by clioquinol. The WB assay of cell cycle-related proteins (CDK2, p21 and p27) further confirmed that S phase arrest is positively correlated with intracellular calcium elevation, which was due to the alterations of the mRNA and protein levels of calcium pumps (SERCA and SPCA). Taken together, these data indicate that clioquinol regulates the level of intracellular calcium ions to induce S-phase cell cycle arrest in human SH-SY5Y cells. Our results demonstrate for the first time that an increase of intracellular calcium content is one of the mechanisms of clioquinol in the inhibition of human neurotypic SHSY-5Y cells.

Randomised controlled trial of WISENSE, a real-time quality improving system for monitoring blind spots during esophagogastroduodenoscopy.

OBJECTIVE: Esophagogastroduodenoscopy (EGD) is the pivotal procedure in the diagnosis of upper gastrointestinal lesions. However, there are significant variations in EGD performance among endoscopists, impairing the discovery rate of gastric cancers and precursor lesions. The aim of this study was to construct a real-time quality improving system, WISENSE, to monitor blind spots, time the procedure and automatically generate photodocumentation during EGD and thus raise the quality of everyday endoscopy. DESIGN: WISENSE system was developed using the methods of deep convolutional neural networks and deep reinforcement learning. Patients referred because of health examination, symptoms, surveillance were recruited from Renmin hospital of Wuhan University. Enrolled patients were randomly assigned to groups that underwent EGD with or without the assistance of WISENSE. The primary end point was to ascertain if there was a difference in the rate of blind spots between WISENSE-assisted group and the control group. RESULTS: WISENSE monitored blind spots with an accuracy of 90.40% in real EGD videos. A total of 324 patients were recruited and randomised. 153 and 150 patients were analysed in the WISENSE and control group, respectively. Blind spot rate was lower in WISENSE group compared with the control (5.86% vs 22.46%, p<0.001), and the mean difference was -15.39% (95% CI -19.23 to -11.54). There was no significant adverse event. CONCLUSIONS: WISENSE significantly reduced blind spot rate of EGD procedure and could be used to improve the quality of everyday endoscopy. TRIAL REGISTRATION NUMBER: ChiCTR1800014809; Results.

Human epidermal growth factor receptor 2, epidermal growth factor receptor, and c-MET overexpression and survival in biliary tract cancer: A meta-analysis.

BACKGROUND: Tyrosine kinase growth factor receptors (TKGFRs) play an important role in the progression of cancer. A variety of studies have investigated the clinicopathologic correlation of these receptors and their influences on patient survival in different types of cancer. As the members of TKGFRs, the biomarkers c-MET, epidermal growth factor receptor (EGFR), and human epidermal growth factor receptor 2 (HER-2) have been extensively investigated in biliary tract cancer (BTC). However, their prognostic value is still controversial. Our study aimed to evaluate the prognostic significance of the three markers in BTC patients based on the published studies. The correlation between high expression of these markers and clinical parameters or overall survival (OS) has been assumed in this paper. MATERIALS AND METHODS: Including PubMed, EMBASE, China National Knowledge Infrastructure, and Springer, a comprehensive search for the related literature published in Chinese and English has been done. Finally, 31 studies were selected in our research. RESULTS: Surprisingly, the meta-analysis indicated that HER-2 high expression was not correlated with age, gender, primary tumor, tumor node metastasis (TNM) stage, lymph node status, and differentiation. We also found that EGFR high expression was not associated with the parameters, such as age, gender, TNM stage, differentiation, or lymph node status. c-MET high-expression was not associated with age, differentiation, gender, TNM stage, or lymph node status. In addition, our study showed that HER-2, EGFR, and c-MET high expression had an adverse influence on OS in BTC, the pooled hazard ratio for HER-2, EGFR, and c-MET was statistically significant. CONCLUSION: The present meta-analysis indicated that EGFR and HER-2 high expression have little impact on OS in patients with BTC while c-MET high expression influenced OS in patients with BTC to a large extent. However, c-MET, EGFR, and HER-2 expression did not show any correlation with those clinical parameters. c-MET may be a potential therapeutic target for BTC.

Imaging of Colorectal Cancers Using Activatable Nanoprobes with Second Near-Infrared Window Emission.

Fluorescent probes in the second near-infrared window (NIR-II) allow high-resolution bioimaging with deep-tissue penetration. However, existing NIR-II materials often have poor signal-to-background ratios because of the lack of target specificity. Herein, an activatable NIR-II nanoprobe for visualizing colorectal cancers was devised. This designed probe displays H2 S-activated ratiometric fluorescence and light-up NIR-II emission at 900-1300 nm. By using this activatable and target specific probe for deep-tissue imaging of H2 S-rich colon cancer cells, accurate identification of colorectal tumors in animal models were performed. It is anticipated that the development of activatable NIR-II probes will find widespread applications in biological and clinical systems.

Synergistic antitumor effect of puerarin combined with 5-fluorouracil on gastric carcinoma.

Combination chemotherapy is a crucial method in the treatment of gastric cancer. The aim of the present study was to investigate the inhibitory effects of puerarin and 5­fluorouracil (5­FU) on BGC­823 gastric cancer cells in vitro and in vivo. The in vitro growth inhibition of puerarin or 5­FU alone or combined on BGC­823 cells was determined using a cell counting kit 8 (CCK­8) on living cells. Apoptotic morphological features and proteins expression levels were detected by Hoechst 33258 staining, an Annexin V/propidium iodide apoptosis kit and western blot analysis, respectively. Tumor xenografts were established in nude mice and the inhibitory effects and side effects were detected. Results of the CCK­8, Hoechst 33258 staining and flow cytometry revealed that the combined treatment was more effective than the separate treatments. The tumor volume was 90.65% of that of the controls and the mean tumor weight was only 0.125 g at the end of the experiment in the combination group compared with the control group (0.822 g). In addition, it was determined that liver and renal toxicity did not increase in combined treatment. These findings showed that puerarin and 5­FU produced a significant synergic effect on gastric cancer cells, while there was no increase in side effects.

XPD Asp312Asn polymorphism and esophageal cancer risk: an update meta-analysis based on 3928 cases and 6012 controls.

BACKGROUND: Although xeroderma pigmentosum group D (XPD) was reported to be related with esophageal cancer (EC) risk, the results remained inconsistent. The aim of this meta-analysis was to make a more precise estimation of the relationship between XPD Asp312Asn polymorphism and EC risk. METHODS: We searched PubMed, Web of Science, Embase, Medline, CNKI and Chinese Biomedical database, covering all publications (up to May, 2014). Statistical analyses were performed with Stata software (version 12.0, USA) and RevMan 5.1 (Copenhagen, 2008). The calculation of odds ratios (ORs) with 95% confidence intervals (CI) was calculated to assess the strength of the association. RESULTS: A total of 15 case-control studies from 13 literatures including 3928 cases and 6012 controls described Asp312Asn genotypes and EC risk. A significant association between XPD Asp312Asn polymorphism and EC risk was found when all the eligible studies were pooled into this meta-analysis. It's also the same result in subgroup analysis of smokers in dominant model (OR=1.63, 95% CI: 1.06-2.50, P=0.03). However, in the stratified analysis by ethnicity and source of population controls, no association between them was discovered. CONCLUSION: The XPD Asp312Asn polymorphism was proved to contribute to the risk of EC in this meta-analysis. Data showed that tobacco consumption may increase the susceptibility of EC.

EBP50 regulates the apoptosis of pancreatic cancer cells by decreasing the expression levels of Bcl-2.

Increasing evidence has demonstrated that ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is involved in the malignant transformation of numerous human cancers. The present study investigated the involvement of EBP50 overexpression in the tumorigenicity of pancreatic cancer (PC). The results revealed that overexpression of EBP50 suppressed cell growth, promoted cell apoptosis and arrested G1-to-S phase progression in two human PC cell lines. Overexpression of EBP50 also suppressed B-cell lymphoma 2 (Bcl-2) expression. Furthermore, nude mouse tumor xenograft models were established by the subcutaneous injection of cell lines stably transfected with an EBP50-expressing plasmid. The in vivo data indicated that overexpression of EBP50 inhibited the growth of the PC tumors and induced cell apoptosis. Thus, the present study demonstrated that EBP50 overexpression induces growth inhibition and apoptosis in PC by decreasing Bcl-2 expression. The results suggest that EBP50 may function as a potential tumor suppressor in vivo and in vitro.

Simple patterned nanofiber scaffolds and its enhanced performance in immunoassay.

Cancer has become the leading cause of death worldwide; early diagnosis and treatment of cancers is critical for the survival of the patients. The concentration of cancer markers in easy-to-access biological fluids can provide great assistance in screening for occult primary cancers, distinguishing malignant from benign findings, determining prognosis and prediction for cancer patients. The multiplex detection technology of a panel of cancer markers can greatly increase the accuracy of disease diagnosis. Herein, we briefly fabricate a high-throughput micro-immunoassay based on the electrospun polystyrene (PS) substrates to improve detection sensitivity. The immunoassay was evaluated by analyzing three different cancer biomarkers (AFP, CEA, VEGF). For AFP, CEA, VEGF immunofluorescence assay, the LOD of assay conducted on electrospun PS substrates before or after plasma and the conventional PS substrates were 0.42, 0.10, 1.12 ng/mL, 0.57, 0.09, 1.24 ng/mL, and 159.75, 26.19, 385.59 pg/mL, respectively (P < 0.05). Due to the high porosity and large surface area-to-volume ratio which is the foremost merit of nanostructures, and the plasma treatment which make the hydrophobic PS nanofibers hydropholic, the nanofibers substrates showed sufficient retention of immunoassay functionality and high potential for capture molecules immobilization. Consequently, the immunofluorescence assay conducted on electrospun PS substrates could significantly enhance the sensitivity and limits of detection.

Clinical significance of EBP50 overexpression assessed by quantum dot analysis in gastric cancer.

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a postsynaptic density-95/disc-large/zonula occludens-1 (PDZ) homologous domain-containing protein that is involved in cell signaling. EBP50 regulates cell apoptosis, proliferation and invasion. In the present study, the prognostic impact factor of EBP50 expression was evaluated using a quantum dot (QD)-based assay and immunohistochemistry (IHC). The EBP50 protein expression in gastric cancer (GC) tissues was evaluated using IHC and QD-IHC. The study included 101 patients with GC (29 females and 72 males, aged 24-81 years), diagnosed and treated at the General Surgery Department of Renmin Hospital of Wuhan University (Wuhan, China) between 2000 and 2005. The survival rate was calculated using the Kaplan-Meier method and log-rank tests. IHC and QD analyses of 101 GC tissue specimens revealed that EBP50-positive tumor cells were frequently present in GC. Increased EBP50 immunostaining was observed in 63 specimens (62.4%). The EBP50 expression levels were correlated with increased tumor size and the male gender. EBP50 was well distributed in the cytoplasm and nuclei of the GC cells. However, EBP50 protein expression exhibited no correlation with age, differentiation, stage or lymph node metastasis. There were no associations between the expression of EBP50 and the mean survival rates (IHC, 50.5 vs. 58.1 months, P>0.05; QD, 55.4 vs. 63.2 months, P>0.05). These findings suggest that EBP50 protein expression is not correlated with the prognosis of patients with GC. QD-IHC and IHC have similar advantages for the detection of EBP50 protein expression.

The detection of EBP50 expression using quantum dot immunohistochemistry in pancreatic cancer tissue and down-regulated EBP50 effect on PC-2 cells.

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a putative tumor suppressor that is correlated with many human cancers. However, the function of EBP50 in pancreatic cancer (PC) has not been described. In this paper, the EBP50 expression level in PC tissues was characterized. In vitro, the effects of EBP50 down-regulation by siRNA in PC-2 and MiaPaCa-2 cells were evaluated. In addition, possible mechanisms that mediate the influence of EBP50 were examined. Our results show that the EBP50 expression pattern changes during transformation as there is a loss of the normal apical membrane distribution and an ectopic cytoplasmic over-expression of EBP50; furthermore, the EBP50 expression level is subsequently decreased during malignant progression. Down-regulation of EBP50 promoted cancer cell proliferation, increased the colony-forming ability of cells and accelerated the G1-to-S progression. Additionally, the loss of EBP50 accentuated ß-catenin activity, increased cyclin E and phosphorylated Rb expression, and attenuated p27 expression compared to control cells. Our results suggest that EBP50 may function as a potential tumor suppressor.

EBP50 gene transfection promotes 5-fluorouracil-induced apoptosis in gastric cancer cells through Bax- and Bcl-2-triggered mitochondrial pathways.

5-Fluorouracil (5-FU) plays an important role in the chemotherapy of advanced gastric cancer. However, genetic factors that affect therapeutic efficacy of 5-FU warrant further investigation. In the present study, using stable transfection of the ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) gene, we explored the genetic influences on 5-FU-induced apoptosis of human gastric cancer cells. Stable overexpression of the EBP50 gene was determined by reverse transcription polymerase chain reaction (RT-PCR) assay and western blot analysis. After treatment with 5-FU, cell growth activities in vitro were investigated by MTT assay. Cell apoptosis was evaluated by Hoechst 33258 staining and flow cytometry of Annexin V-FITC/PI staining. Compared with the BGC823 or BGC823/neo cells, EBP50 mRNA and protein levels in the BGC823/EBP50 cells (EBP50-transfected BGC823 cells) were markedly higher. Chemosensitivity and apoptosis rates of the BGC823/EBP50 cells were higher compared to the BGC823 and BGC823/neo cells following treatment with 5-FU. Stable overexpression of extrinsic EBP50 distinctly increases the 5-FU-induced apoptosis of gastric cancer cells, and is a novel strategy by which to improve the chemosensitivity of gastric cancer to 5-FU.

Thymoquinone inhibits growth and augments 5-fluorouracil-induced apoptosis in gastric cancer cells both in vitro and in vivo.

Thymoquinone (TQ), a component derived from the bioactive constituent of black seed (Nigella sativa), has been shown to exert biological activity on various types of human cancers. However, there are few studies addressing its effects on gastric cancer. Here, we present the first report describing the chemosensitizing effect of thymoquinone and 5-fluorouracil (5-FU) on gastric cancer cells both in vitro and in vivo. Studies have shown that pretreatment with TQ significantly increased the apoptotic effects induced by 5-FU in gastric cancer cell lines in vitro. Moreover, we found that TQ enhanced the 5-FU-induced killing of gastric cancer cells by mediating the downregulation of the anti-apoptotic protein bcl-2, the upregulation of the pro-apoptotic protein bax, and the activation of both caspase-3 and caspase-9. In addition to the in vitro results, it has been shown that the combined treatment of TQ with 5-FU represents a significantly more effective antitumor agent than either agent alone in a xenograft tumor mouse model. These data suggest that the TQ/5-FU combined treatment induces apoptosis by enhancing the activation of both caspase-3 and caspase-9 in gastric cancer cells. These results, which provide molecular evidence both in vitro and in vivo, support our conclusion that thymoquinone can activate caspase-3 and caspase-9 and thus result in the chemosensitisation of gastric cancer cells to 5-FU-induced cell death.